GB2218101A - TNF-alpha inhibitors - Google Patents

Abstract

A protein having a selective tumour necrosis factor (TNF)- alpha inhibitory activity, but which does not block other proteins, particularly IL-1, is prepared by extraction from urine of febule patients.

Description

1 BIOLOGICALLY ACTIVE PROTEINS The present invention relates to a novel

protein having an inhibitory effect against Tumour Necrosis Factor a-mediated activity, to the isolation and purification of such a protein from natural sources, to its preparation by DNA manipulation and to the use of such a protein in the treatment of conditions associated with excessive or unregulated TNFa production.

Tumour necrosis factor (TNF) is an activity embodied by a family of at least two proteins, a and P, which are cytotoxic for tumour cells and inhibit their growth in culture [E. Carswell et. al. "An endatoxininduced serum factor that causes necrosis of tumours", Proc. Natl- Aced. Sci. USA, 72, p3666 (1975)]. Tumour necrosis factor a (TNFa), also termed "cachectin", is mainly produced by cells of the monocyte/macrophage lineage in response to "stress" signals which accompany invasive stimuli such as bacteria, viruses, tumoups and other toxins. TNFp, commonly termed Illymphotoxin", is mainly Qroduced by lymphoid-cells. TNFp has many activities similar to those of TNFa, but it appears to be less potent although this may be as a result of difficulties in preparing pure TNFP.

TNFa mediates and participates in a wide range of biological activities [B. Beutler et. al., "Identity of tumour necrosis factor and the macrophage-secreted factor cachectin", Nature, 316, p552 (1985)] sharing several of them with interleukin 1 (IL-1) [0. Le et.

al., "Tumour necrosis factor and interleukin I: cytokines with multiple overlapping biological activities", Laboratory Invest., 56, p234 (1987)]. Elevated levels of TNFa induced by, for example, tumour cells may lead to weight loss and cachexia and TNFa has also been implicated as a principal mediator of endotoxic shock (septic shock) which can be fatal. Other biological effects of TNFa include hypotension, fever (induced by stimulation of hypothalamic prostoglandin E2 (PGE2) synthesis), coagulopathy-(induced by stimulation of vascular endothelial cells which release, for example, tissue factor) and tissue destruction (induced by, for example, stimulation of a series of proteinases, including collagenase production by dermal fibroblasts and synovial cells) [C. Dinarello et. 1-, "Tumour necrosis factor (cachectin) is an endogenous pyrogen and induces production of interleukin 111, J. Exp. Med. , 163, p1433 (1986); J. Dayer et. al., "Cachectin/tumour necrosis factor stimulate collagenase and prostaglandin E2 production by human dermal fibroblasts and synovial cells", J. Exp. Med., 162, p2163 (1985)1.

There exists, therefore, a need to develop a cachectin/TNFa inhibitor which prevents endotoxic shock. cachexia and the other deleterious effects described above. It has been shown that passive immunisation of animals against cachectin can prevent endotoxin-induced death, mediated by TNFa antibodies [B. Beutler et.

al., Nature, 316, supral.

We have now identified a novel protein which has a patent inhibitory effect against TNFa-mediated activities without significant concomitant inhibition of IL-1-mediated activity. The protein is hereinafter identified as Tumour Necrosis Factor a Inhibitor (TNFa INH).

Thus in one aspect of the invention, we provide a protein which selectively inhibits tumour necrosis factor a-mediated activity.

4 3 - 1 As used herein, selective inhibition as shown by the inhibitor of the invention.is identified as the ability to block TNF-mediated activity while lacking the ability to block other proteins which have in common with TNF certain but not all of the biological activities of TNF, such as IL-I.

Preferably, the tumour necrosis factor a inhibitor of the invention is in a substantially homogeneous form,..h.!ing substantially free from major contaminents and/or substantially free from other proteinaceous material.

The tumour necrosis factor a inhibitor according to the invention has been found to have one or more of the following characteristics:

(a) a molecular weight in the range 40 to 60 kDa, determined by molecular sieve chromatography; - (b) an iso-electric point (pI) in the range 5.5 to 6.1, determined by chromatofocussing; (c) inhibition of the standard TNF assay of differential cytotoxicity for murine L929 cells treated with actinomycin D,. as described by G. Nedwin et. al. "Effects of interleukin 2, interferon-y and mitogens on the production of tumour necrosis factors a and P", J.

Immunol., 135, p2492 (1985). This inhibition can be overcome by further addition of TNFa, indicating that the inhibition is competitive. The inhibitor is also an inhibitor of TNFp activity, although inhibition of TNFa in this assay is more efficient than that of TNFp; (d) inhibition of TNF-induced PGE2 release from human fibroblasts and synovial cells; (e) the inhibitor interferes with the binding of TNFi to U937 cells (a monacytic tumour line) as evidenced by inhibition of binding of radiolabelled TNFa (125 I-TNFa); - (f) the dissociation of a pre-formed TNFa: U937 cell complex is promoted by the inhibitor in a temperature dependent manner; (9) the inhibitor does not degrade TNF by proteolytic cleavage; (h) the inhibitor does not inhibit IL-1 receptor-binding activity e.g. the binding of radiolabelled IL-1 (13 I-IL-1a) to the murine thymoma subline EL4-6.1.

We have found that the protein of the invention when further purified has a molecular weight of about 33000 daltons as determined by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE).

There is thus provided as a further or alternative aspect, a protein which selectively inhibits TNFa-mediated activity which has one or more of the following characteristics:

(a) a molecular weight of about 33 kDa, determined by SDS PAGE; (b) an iso-electric point (pI) in the range 5.5 to 6.1, determined by chromatofocussing; (c) inhibition of the standard TNF assay of differential cytotoxicity for murine L929 cells treated with actinomycin D, as.described by G. Nedwin et. al. "Effects of interleukin 2, interferon-y 25 and mitagens on the production of tumour necrosis factors a and 0", J. Immunol., 135, p2492 (1985). This inhibition can be overcome by further addition of TNFa, indicating that the inhibition is competitive. The inhibitor is also an inhibitor of TNFP activity, although inhibition of TNFa in this essay is more efficient than that of TWp; (d) inhibition of TNF-induced PG release from human fibroblasts and synovial cells; (e) the inhibitor interferes with the binding of TNFa to U937 cells (a monocytic tumour line) as evidenced by inhibition of binding of radiolabelled TNFa (125 I-TNFa (f) the dissociation of a pre-formed TNFa: U937 cell complex is promoted by the inhibitor in a temperature dependent manner; (g) the inhibitor does not degrade TNF by proteolytic cleavage; (h) the inhibitor does not inhibit IL-1 receptor-binding activity e.g. the binding of radiolabelled IL-1 (123 I-IL-1a) to the murine thymoma subline EL4-6.1.

Preferably the TNFa INH of the present invention has both of the characteristics (a) and (b) and one or more of the characteristics (c) to (h).

In particular, the TNFa INH of the present invention has all of the characteristics (a) to (h).

The protein of the invention has an amkno terminal amino acid sequence as follows:

Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile-His- Pro-G1n-Cys-Asn-Ser-Ile It is further believed that the next three amino acids provide a glycosylation site and that the sequence thus continues Asn-Ser-Thr-Lys.

1 1 It 6 - It will be appreciated that a TNFa inhibitor according to the invention will comprise an amino acid sequence substantially corresponding to the sequence of native TNFa INH and containing an amino terminal sequence substantially identical to that described above. The sequence of a TNFa inhibitor according to the invention will thus be identical to the sequence of native TNFa INH or contain one or more deletions, substitutions, insert.tons,".;.nversions or additions of allelic origin or otherwise, the resulting sequence will have at least 80% and preferably 90%, homology with the sequence of native TNFa INH and retain essentially the same biological properties of the protein.

The TNFa inhibitor of the invention has been demonstrated to be proteinaceous in that it is inactivated by heating in a time and temperature dependent manner is is destroyed by treatment with trypsin or pronase.

The TNFa INH of the invention has also been shown to be a glycoprotein since treatment with the enzyme Endoglycosidase F reduces the molecular weight by 7 to 8 kDa.

In a further or alternative aspect of the invention there is thus provided a Wa inhibitor as defined herein, but which is in a substantially unglycosylated state.

The inhibitors of the invention are of interest in the treatment of conditions in which it is desirable to inhibit Wa activity, for example, those which arise from the effects of TNFa such as weight loss, shock, cachexia and chronic local inflammation, rheumatoid arthritis, disseminated intravascular coagulation and nephritis.

Q 7 There is thus provided as a further aspect of the invention TNF'a inhibitor as herein defined or a pharmaceutically acceptable derivative thereof for use as an active therapeutic agent, in particular, in the treatment of conditions associated with excessive or unregulated TNFa production.

In a further or alternative aspect of the invention there is provided a method for the treatment of conditionn associated with excessive or unregulated TNFa production in a mammal including man comprising administration of an effective amount of a TNFa inhibitor 10 as herein defined or a pharmaceutically acceptable derivative thereof.

There is also provided in a further or alternative aspect of the invention use of a TNFa inhibitor as herein defined or a pharmaceutically acceptable derivative thereof for the manufacture of a medicament for the treatment of conditions associated with excessive or unregulated TNFa production.

It will be appreciated by those skilled in the art that reference herein to treatment extends to prophylaxis as well as the treatment of established conditions or symptoms.

It will be further appreciated that the amount of TNFa inhibitor of the invention required for use in treatment will vary not only with the route of administration but also with the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or 25 veterinarian. In general however, a suitable dose will be in the range of from about 5.0 to 500 ig per kilogram of bodyweight per day, Z1 for example, in the range 30 to 300ig/kg/day, preferably in the range 50 to 1504g/kg/day.

The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three,. four or more sub-doses per day.

While it may be possible that, for use in therapy, a TNFa inhibitor of the invention may be administered as the raw protein it is preferable to present the active protein as a pharmaceutical formulation.

The invention further provides a pharmaceutical formulation comprising a TNFa inhibitor as herein defined or a pharmaceutically acceptable derivative thereof together with one or more pharmaceutically acceptable carriers thereof and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be 'acceptable' in the sense of being compatible with the ingredients of the formulation and not deleterious to the recipient thereof.

The inhibitors according to the invention may therefore be formulated for parenteral administration (e.g. by injection, for example bolus injection or continuous infusion) and may be presented in unit dose form in ampoules, pre-filled syringes, small volume infusions or in multi-dose containers with an added preservative. The compositions may take such forms as suspensions or solutions in aqueous vehicles, and may contain formulatopy agents such as suspending, stabilising and/or dispersing agents. Alternatively, the active ingredient may be in powder form obtained by aseptic isolation of sterile solid or by Iyophilisation from solution, for constitution Z Z 9 with a suitable vehicle, e.g. sterile, pyragen-free water, befare USPS.

The T,%F3: inhibitor of the invention may also be used in combination with other therapeutic agents, for example, other cytokines or inhibitors thereof.

The invention thus provides, in a further aspect, a combirtation. comprising a TNFa inhibitor as herein defined or.z pharmaceutically acceptable derivative thereof together with another therapeutically active agent, for example, other cytokines or inhibitors thereof- The proteins of the invention may be prepared by purification.

from natural sources and, where appropriate followed by chemical modification, or they may be prepared by conventional methods known in the art for the preparation of proteins, for example, by recombin.an;t DNA techniques.

Accordino to a further aspect of the present invention, there is provided a process for producing the tumour necrosis factor a inhibitor of the invention by purification from natural sources, particularly the urine of human febrile patients. Such purificatiori, for example, comprises the steps of concentrating the crude urirTe- of febrile human patients, precipitating crude TNFa INH from the UrIne and fractionating the TNFa INH from the other proteins of this precipitate by one or more of, for example, ion exchange column chromatography, gel filtration chromatography, hydrophobicity chromatography, immunoabsorption and affinity chromatography on immobilized TNFa.

The tumour necrosis factor a inhibitor of the invention. is also obtainabLe from macrophage containing hu-nan tissue, for example, - 13 lung lavages and extracts of human liver, from which it may be obtained by standard purification techniques such as those described above.

Natural and recombinant TNFa INH produced according to the procedures described herein may be purified through a series of steps as listed above. After each of the purification steps, the presence and purity of the TNFa INH may be meesured in an assay of cytotoxicity in the presence of actinomycin D (acti D) using a TNF-susceptible cell line L929, as described by G. Nedwin et al., J. Immunol., 135, loc.

cit.

In a preferred embodiment of the process the TNFa INH is initially isolated from untreated urine collected from febrile human patients (>38. 5 0 C) devoid of urinary infections using a standard concentration technique, for example, ultrafiltration. A crude fraction may then be precipitated from the crude urine using ammonium sulphate e.g. by addition of ammonium sulphate to a concentration of 805o (w/v) at 40C with stirring. Preferably the ammonium sulphate may be added in a stepwise manner and material precipitated at lower concentrations e.g. at 40% (w/v) discarded. The ammonium sulphate may be removed by dialysis and the resulting fraction purified to separate the TNFa INH from other proteins by a variety of chromatographic methods.

Thus, the TNFa INH concentrate may be purified by ion-exchange chromatography which separates proteins according to their differences in electrical charge, which is a reflection of the acid/base properties of the proteins. Suitable materials for anion-exchange chromatography include aminoethylcellulose derivatives, for example, - 11 quaternary-aminoethyloellulose (QAE-cellulose) or diethylaminoethylcellulose (DEAE-cell-lullose) which are widely commercially available. The anion-exchange column should be equilibrated prior to applying the concentrate using a suitable buffer, such as TrisHCI, optionally containing a chelating agent such as EDTA. Bound material may be eluted from the column using a salt solution (for example, 0.8M sodium chloride made up with the equilibration buffer).

The pooled active fractions from the anion-exchange chromatography. Suitable materials for cation-exchange chromatography include derivatives of cellulose such as carboxymethyl (CM) cellulose or Sulphopropyl Sepharose (Pharmacia, Uppsala, Sweden). The column should be equilibrated with a suitable buffer, such as sodium acetate and bound material may be eluted with the equilibration buffer containing, for example, 0.5M sodium chloride.

The pooled active fractions are further purified by affinity chromatography on bound recombinant human TNFa (rhTNFa), coupled to a suitable matrix, for example, Mini-Leak Agarose (Kem En Tec, Biotechnology Corp., Denmark). The column should be buffered using, for instance, a phosphate buffer (e.g. 0.8M potassium phosphate pH 20 8-6). Active groups not bound to rhTNFa should be blocked usinG ethanolamine- HCI pH 8.5 buffer. The column should be equilibrated with a suitable buffer, for example, Tris-HCl optionally containing sodium chloride and the TNFa INH eluted with an acidic (pH 3.5) glycine buffer. The eluted fractions should immediately be balanced 25 to pH 7.0 by the addition of, for example, Tris base.

The active pooled fractions are preferably Iyophilised prior to the final purification step of reverse-phase FPLC (fast protein liquid -s application to the FPLC column, the chromatography). Prior to it Iyophilised TNFa INH fpaction should be buffered with an appropriate buffer such as trifluoroacetic acid (TFA'j (Fulka, Buchs, Switzerland), heptafluorobutyric acid (HFBA) or acetic acid. Elution of the TNFa INH from the FPLC column may be carried out using conventional techniques, for example, made up with a suitable buffer as previously described, optionally containing an alcohol, stance, N-propenol.

The eluted fraction should immediately be buffered with, for example, ammonium bicarbonate and lyophilised. The TNFa INH is now in a substantially homogenous form suitable for further assaying of biological activity and for preparation in an appropriate form for therapeutic use.

There is thus provided as a further or alternative aspect of the invention a protein which selectively inhibits TNFa-mediated activity which is substantially identical to that obtained in the above process.

The ability to purify TNFa INH of the present invention to homogeneity has enabled the sequencing of the N-terminal portion of this protein molecule. This sequence will assist in the cloning of a gene coding for the TNFa INH thus enabling the production of large quantities of TNFa INH in pure form for further biological investigation and eventually for therapeutic testing and use.

Samples of the homogeneous TNFa INH of the present invention can be sequenced by conventional techniques, for example, using a commercially available automated sequencer employing either ninhydrin or gas phase detection. The first 17 residues of the amino terminal.

- 13 portion of the human TNFa INH as herein defined comprises the following sequence:

Asp-Ser-Val-Cys-Pro-Gln-Gly-Lys-Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser-I (identified using an automated sequencer, Model 477A from Applied Biosystems).

It is further believed that the next three amino acids provide a glycosylation site and that the sequence thus cont.,.nues Asn-Ser-Thr-Lys.

It will be appreciated that one potential method of providing large quantities of TNFa INH in pure form is through recombinent'DNA techniques which are well known in the art. However, the successful use of such techniques requires not only that the natural and recombinant TNFa INH or its activity be accurately measured, but also that both the natural and recombinant product be purifiable to homogeneity.

According to yet another aspect of the present invention, there is provided a process for producing a tumour necrosis factor inhibitor of the invention or a derivative thereof by the expression of a DNA sequence encoding for such an inhibitor in a suitable transformed host. Such a process involves the culturing of a host transformed with recombinant DNA molecules comprising DNA sequences encoding the inhibitor which have been inserted into a suitable vector.

Suitable eukaryotic and prokaryotic hosts may be, for example, strains of bacteria, yeasts, other fungi, and animal cells (includino 25 insect cells) and plant cells in tissue. Particularly preferred host le cells are yeaet cells, E. coli cells and animal cells.

Expression of a protein having tumour necrosis fac'l-,De inhibitor activity is achieved by culture of the transformed host cells in a suitable growth medium. Normally such a medium will contain a source of nitrogen such as ammonium sulphate, a source of carbon and energy such as glucose or glycerol, trace elements and factors essential to growth of the particular cells. The precise culture conditions will be dependent upon the chosen host; thus, for example, in the case of E. coli submerged aerobic fermentation is preferred, preferably at about 370C.

In addition, expression may be induced, for example, by the addition of an inducer or the use of inducing conditions for the promoter system being used in the expression vector.

Depending upon the host, the TNFa inhibitor may be produced as granular inclusion bodies which can be recovered, after cell lysis, by differential centrifugation; these can be solubilised by conventional methods and purified by the methods described herein for purification of urinary TNFa INH. Alternatively, the TNFa inhibitor may be in solution in the cytosol, secreted into the periplasmic space or conveniently secreted into the culture medium.

The host cells are transformed by recombinant DNA molecules which comprise a DNA sequence encoding for a TNFa inhibitor which has been inserted into an expression vector.

Such expression vectors may consist of segments of chromosomal, non-chromosomal and synthetic DNA sequences, such as various known derivatives of SV-40 and known bacterial plesmids, for example, "natural" plasmids such as ColEl, pSCIOI or pRSF2124 and - 15 phage DNAs, or ",artificial" plasmids (constructed in vilt-ro) such as pBR322, prIB9 or pAT153. Phage DNAs include, for example, the iiineTJUS derivatives of phage lambda and other DNA phages, for example M13, and other filamentous single-stranded DNA phages. Vectors useful in yeasts include the 2[1 plasmid, and those useful in eukaryotic cells such as animal cells include those containing SV-40 adenovirus and retrovirus.

Such expression vectors may also be characterised by at least one expression control sequence which may be operatively linked to the TNF inhibitor DNA sequence such that it controls and regulates the expression of the cloned DNA sequence. Examples of useful expression control sequences include the lac, trp, tac and trc systems, major operator and promoter regions of phage X (such as the PL Promoter under the control of the thermolabile ts cI857 repressor), the control region of fd coat protein, the glycolytic promoters of yeast (e.g. the promoter for 3-phosphoglycerate kinase), the promoters of yeast acid phosphatase (e.g. Pho 5), the promoters of yeast a-mating factors, and promoters derived from polyama, adenovirus, retrovirus, and simian virus.

In addition, such expression vectors may possess various sites for insertion of the TNFa inhibitor DNA sequences of this invention. These sites are characterised by the specific restriction endonuclease which cleaves them. Such cleavage sites are well recognised by those skilled in the art. The expression vector, and in particular the site chosen therein for insertion of a selected DNA fragment and its operative 'linking to an expression control sequence, is determined by a variety of factors including the number of sites susceptible to a - 16 given restriction enzyme, the size of the protein to be expressed, contamination or binding of the protein to be expiessed by host cell proteins which may be difficult to remove during purification, the location of start/stop codons, and other factors recognised by those skilled in the art. Thus the choice of a vector and insertion site for a DNA sequence is determined by a balance of these factors, not all selections being equally effective for a giveli.11. case.

Likewise, not all host/vector combinations will function with equal efficiency in expressing the DNA sequences of this invention.

The selection is made, depending upon a variety of factors including compatability of the host and vector, ease ol recovery of the desired protein, expression characteristics of the DNA sequences and the expression control sequences operatively linked to them, or any necessary post-expression modifications of the desired protein.

The DNA sequences of the invention which on expression code for proteins with TNFa inhibitor activity may be isolated by screening various DNA libraries for such DNA sequences using a series of DNA probes. The DNA probes may be prepared from the purified natural protein which is used as a source of amino acid sequence data. The purified natural protein may be prepared, for example, from febrile human urine as described above. Degenerate DNA sequences coding for various portions and fragments of the amino acid sequence, e.g. in combination with Lathe probes, are used to design the DNA probes.

Thus, various DNA libraries are screened for DNA sequences coding for the TNFa inhibitors of the invention. Such libraries include chromosomal gene banks and cDNA or DNA libraries prepared from cell lines or tissue that are demonstrated to produce TNFa 17 inhibitors, such as alveolar macrophages or liver tissue. Screening ma% be by dire--' immune expression, for example in Xatll or similar systems, or, in the case where a TNFa INH producing cell is identified, by identification of TNFa INH specific rrRNA by direct expression in Xenopus oocytes.

A variety of conventional cloning and selection techniques may be used to locate and identify DNA sequences '.--.%-----'--ncode on expression in an appropriate eukaryotic or prokaryotic host for the TNFa inhibitors of this invention. These selected DNA sequences may themselves be used as probes to select other DNA sequences coding for TNFa inhibitors or may be used in appropriate recombinant DNA molecules to transform appropriate eukaryotic or prokcaryotic hosts for the production of TNFa INH encoded by them.

The invention includes within its scope single and double stranded DNA sequences encoding for TNFa INHs, vectors containing such sequences suitable for transformation of a host organism and host cells transformed with such DNA sequences.

According to a further aspect of the present invention we provide a protein with selective TNFa inhibitor activity produced by expression of a host transformed with a DNA sequence encoding for such a TNFa inhibitor protein. TNF inhibitors of the invention which are prepared by the expression of a DNA sequence encoding such inhibitors in a transformed host will thus be identical to the sequence of native TNFa INH or contain one or more deletions, substitutions, insertions, inversions or additions of allelic origin or otherwise, the resulting sequence will have at least 8.011'0 and preferably 90'10 homology with the sequence of native TNFa INH end retain essentially the same biological 1:t 1 - prone-tip_S. in particular, a TNF inhibitor of the invenMon may include an Nterininal methionine. Also, for example, the DNA seouence of the invention coding for TNFa INH-may be fused in an expression vector to a portion of a DNA sequence coding for a eukaryotic or prokaryotic polypeptide to assist the expression of the Wa INH encoding DNA sequence or aid secretion, maturation or purification of the TNFa INH from the host; the fused polypp-ptide_,may be removed intre-or extra-cellularly by known techniques or the INFa INH may be used together with the fused polypeptide.

The TNFa INHs produced by culturing of the eukaryotic and prokaryotic hosts transformed with DNA sequences encoding for Wa INHs can then be employed, after purification, in the pharmaceutical compositions of this invention.

It will be appreciated that, when produced by animal cells, the TNFa INH of the invention will be a glycoprotein. Prokaryotic expression systemswill, however, produce the protein in an unglycosylated state. In addition, the glycosylated protein may be substantially deglycosylated by techniques known in the artY for example, by the use of endoglycosidese enzymes.

The following non-limiting Examples illustrate the invention.

All temperatures are in 'C and all percentage concentrations are i,/v.

TNFa Inhibition Assay The percentage of TNFa INH activity in the fractions described in the Examples was determined by assuming that the optical density (OD) values from murine L929 cells stimulated by actinomycin D (acti D) corresponded to 100,10 inhibition, whereas the OD from cells cultured - 19 1 with actinomycin D and TNFa corresponded to a maximal cell moptality of 0% TNFa inhibition. The TN'ra used in the assa -,-.as recomlbina-it human TNFa (rhTNFa) produced in E. coli as described by A. Marmenout et. al., "Molecular cloning and expression of human tumour necrosis factor and comparison with mouse tumour necrosis factor", Eur. J. Biochem., 152, p515 (1985). Thus the percentage of TNFa inhibition in the assay of cytotoxicity was calculated accord.---g to formula (I) Percentage of TNFa INH activity (OD with acti D + rhTNFa + TNFa INW - (OD with acti D + rhTNFa) -100 X (OD with acti D) - (OD with acti D + rhT%Fa) Example 1

Purification of Urinary TNFa INH a) Concentration of Protein from Human Urine Human urine (15 litres) was freshly obtained from a pool of (I) five patients prior to any treatment. Two of the patients were suffering from small-cell carcinoma, one from malignant histocytosis, one from polymyocitis and one from sepsis. All were highly febrile (>38.50C and devoid of urinary infections. The urine was concentrated at 40 on an Amicon ultrafiltration hollow fibre 25 apparatus, with a molecular size cut-off of about 5 kDa.

b) Precipitation of Protein from Human Urine The concentrated urine pool was satucated with soli4; sulphate by adding the sulphate slowly with constant stirring at 40 until an ammonium sulphate concentration of 40% was reached. The precipitate was removed by centrifugation, discarded, and the supernatant adjusted to 805'0 saturation with addition of further ammonium sulphate. A pellet was obtained by centrifugation which was resuspended in 150 ml of 20 mM sodium phosphate (pH 7.2) and 150 mM sodium chloride. The ammonium sulphate was removed by dialysis at 40 using 10 mM Tris-HCl pH 7.4, 2 mM EDTA and 5 mM benzamidine HCl.

c) Identification of TNFa INH Activity The semi-purified fraction of Example l(b) was tested in a cytoxicity assay with the TNF-susceptible cell-line L929 in the presence of actinomYcin D. At a 1:20 dilution of the semi-purified fraction, total inhibition of the cytotaxic effect induced by rhTNFa was observed so that the ODS7onm value was identical to that measured in the presence of actinomycin D alone (OD 57onm 7- 1.5).

Furthermore, inhibitory activity was observed in dilutions of the fraction of up to 1:160 on cells (0D570nm = 0.83) whereas the control value of rhTNFa at a final concentration of 0.2 ng/ml measured in the presence of actinomycin D was lower (ORS7Onm = 0.73), so that 50% of inhibition was observed at a dilution of approximately 1:100 (OP57onm 1.10).The TNFa INH had no effect an cell viability when tested without actinomycin D.

1 - 2i d) Com.parison of the Effect of T.'Fa-IN,tJ, on TNF a a-d E induced, CVt-DtOXiCity A cytotoxicity assay was conducted using the TNII-susceptible cell-line L929 in the presence of actinomycin D using a range of concentration of TNFa or TNFp to induce the cytotoxic effect. The semi-purified fraction from Example 1(b) was tested at 1:20, 1:50 and 1:80 dilutions. Control tests were performed ir..the absence of the TNFa or TWO cytokine, and in the absence of inhibitor. The results are shown in Table 1, below, and demonstrate that the inhibitor of the invention does have some inhibitory effect on TNFp mediated cytoxicity ranging from approximately 50% down to 2% of the TNFa inhibition with increasing TNFP concentration.

1 - 917 - TABLE 1

7 Final Concentration Orm of of TiNF (a or p) (pg/ml)' Cytokine Added to Actinomycin-D added to Treated L929 cells cells Final Dilj'..-ic)n ol SephaeRyl S-200 Inhibitory Fraction on L929 cells (OD57onm None 1/20 l/50 1/80 0 0 >1.90 >1.90 >1.90>1.90 a --- NP N Q- ND ND p 1.16 1.66 1.46 1.39 cc 1.30 >1.90 1.72 1.68 8 1.02 1.51 1.21 1.22 a 1.02 >1.90 1.72 1.68 8 0.65 1.03 0.84 0.68 a 0.73 1.78 1.69 1.51 0.37 0.71 0.59 0.52 250 0.38 1.70 1.70 1.33 0.24 0.44 0.31 0.24 500 0.30 1.52 1.49 1.11 0.17 0.30 0.26 0.18 1 250 0.19 1.09 1.10 0.76 0.11 0.13 0.19 0.13 2 500 a 0.06 1.03 0.80 0.47 p ND ND ND KID 1 Example _2 Gel Filtration of Urinary TNFa INH The semi-purified TNFa INH of Example l(b) was purified by gel filtration chromatography at 40 on a Sephacryl 5-200 column (0.9 x 60 cm) (Pharmacia, Uppsala, Sweden) equilibrated in 50 akI Tris-HCI buffer (pH7.4) containing 100 W sodium chloride. A sample of the protein fraction (20 mg, 0.8 ml) was applied to the c-llu.m,,and eluted with the same buffer at a flow-rate of 5.4 ml/hr. Fractions (1.35 ml) were collected and tested for TNFa INH activity. The TNFa INH activity eluted from the gel in a s-ingle peak. The inhibitory activity showed an apparent molecular weight of 40 to 60 kDa (see FiguPe 1).

Example 3 Chromatafocussing of Urinary TNFa INH The semi-purified TNFa INH of Example 1(b) was chromatofocussed at 4 0 on a Mono-P pre-packed column (HR 5/20, 5 x 200 mm) (Pharmacia, Uppsala, Sweden equilibrated in 25 m'l Bis-Tris buffer adjusted to pH 7.1 with imidodiacetic acid (Fluka, Buchs, Switzerland). A sample of the protein fraction of Example l(b.) (30 20 mg) was applied to the column and eluted with a polybuffer 74/iminodiecetic acid at pH 4.0. Column fractions (I ml) were tested at 1:10 dilution for their effect in the rhTNFa (0.2 ng/ml) cytotoxicity assay in the presence of actinomycin D (1ig/ml). The actual pH of each column fraction was determined with a pH meter, the 25 bulk of the TNFa INH activity being contained in the eluted fractions pT e-tween pH 5.5 and 6.1 (E=-e Fic-ure 2). Thl I- of the TNFa INH protein.

Example 4 Lon-Exchange Chromatography of Urinary Wa INH The semi-purified TNFa INH of Example 1(b) was purified by anion-exchange chromatography at 40 on a DEAE Sephadex column (2.6 x 20cm) (Pharmacia, Uppsale, Sweden) equilibrated in 1OmM Tris-HCl buffer pH 8.0, containing 2-WI EDTA. Bound material was eluted from the column with the equilibration buffer containing 0.81.1 sodium chloride. Fractions (8.0m1) were collected, tested for TNFa INH activity and the inhibitory fractions were pooled (160m1) and dialysed against 1OMM sodium acetate buffer pH 5.0 (4 x 2 litres).

The TNFa INH was further purified by cation-exchange chromatography at 40 an a Sulphopropyl-Sephadex column (0.8 x 15 cm) (Pharmacia, Uppsala, Sweden) equilibrated in 10M sodium acetate buffer pH 5.0. Bound material was eluted from the column with the equilibration buffer containing 0.5M sodium chloride. Fractions (7.5m1) were collected, tested for TNFa INH activity and the inhibitory fractions were pooled and concentrated 20-fold an an Amicon ultrafiltration apparatus with a molecular size cut-off of about 10 kDa.

Example 5 25 Ge! Filtration of Urinary T.%F: I.NH The TNFa INH concentrate from Example 4 was purified by gel filtration chromatography at 4 0 on a Sephacryl S-200 column (2.6 x 100 - 25 cmlo (PhaEmacia, Uppsala, Sweden) equilibrated with 50mili TTis-,'iU- I ph 7.4 b,.'Fer containing 100mi-I sodiu-n chloride. A sample ot the p7- otein fraction from Example 4 (200mg) was applied to the column and eluted with the equilibration buffer at a flow-rate of 27ml/hour. Fractions (9.Oml) were collected, tested for TNFa INH activity and the inhibitory fractions were pooled. The column was calibrated with dextran blue OB), 2000 kDa; bovine serum albumin (BSA), 67 kDa; ovalbumin (OA), 43 kDa; a-chymotrypsinogen-A (aCT), 25 kDa; and ribonuclease A (RNase), 13.5 kDa, as shown in Figure 4.

Example 6 Affinity Chromatooreohy of Urinary TNFa INH A TNFa affinity column was prepared by coupling recombinant h.uman TNFa (1.Omg.) to Mini Leak Agarose (Kem En Tec, Biotechnology Corp.,. Denmark) in 0.81-11 potassium phosphate buffer pH 8-6. The remaining active groups were blocked by incubation in 0.1M ethanolamine-HIl buffer pH 8.5. The gel was washed with 50mM Tris-HCI pH 7.4 buffer containing -lOOmM sodium chloride (3 x 50ml). A sample of the TNFa INH fractions from Example 5 (15ml) was applied to the colu-nn and e1uted 20 with a 0.2M glycine-HC1 pH 3.5 buffer. Fractions (I.Oml) were collected, immediately adjusted to pH 7.0 by addition of IM Tris (5 to 40Ll) and tested for TNFa INH activity.

Example 7 Reverse Phase FPLC Chromatography of Urinary TNFa INH The TNFa INH fractions from Example 6 were lyophilised, dissolved in 0.1% trifluoroacetic acid (2.0m1) and loaded onto a ProRPC - 26 revezse-phase FPLC column (5 x 20 cm) (Pharmacia, Uppsale, Sweden) equilibrated in 0.11% trifluoroacetic acid. Bound material was eluted with a 0 to 100% acetonitrile gradient in 0.1% trifluoroacetic acid at a flow rate of 0.3m1/minute. To each fraction (0.75m1) 0.5M ammonium bicarbonate (101) was added and the eluted material was lyophilised.

The reverse-phase FPLC chromatography revealed one major peak corresponding to TNFa INH activity. The lyophilised fractions containing this activity were dissolved in 1OmM Tris-HCl pH 7.4 buffer containing 2mM EDTA and analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) using the method described by U. LaemmU et al., Nature, 277, p 680 (1970). The Wa INH was found to elute with a molecular weight of 33 kDa (see Figure 4). Samples run under non-reducing conditions were tested for TNFa INH activity at 1:10 dilution on L929 cells in the presence of 0.15 mg/ml of rhTNFa. The activity directed against rhTNFa migrated with an apparent molecular weight identical to the 33 kDa band on the gel run under reducing conditions.

Example 8 Protein Sequencing of Urinary TNFa INH The TNFa INH fraction isolated from the reverse-phase FPLC chromatography was concentrated in vacuO and spotted onto a conditioned sequencer filter. The protein was analysed with an Applied Biosystems Model 477A protein sequencer. Fractions from the sequencing cycles were evaporated to dryness and resuspended in N,N-diisopropylethylamine acetate and acetonitrile prior to injection into an HPLC column for residue identification.

The first 17 amino acid residues of the N-terminal were identified and have the sequence: Asp-Ser-Val-Cys-Pro-Gln-Gly-LysTyr-Ile-His-Pro-Gln-CysAsn-Ser-Ile. It is further believed that the next three amino acids provide a glycosylation site and that the sequence thus continues Asn-SerThr-Lys. This sequence is not significantly homogeneous to any protein sequence contained in the NBRF Protein Sequence database (November 1988).

Example 9

Demonstration that TNFa INH is a Protein a) Time and Temperature Dependency The Sephacryl S-200 purified TNFa INH of Example 2, obtained by bulking the tubes of the active fractions, was heated at 560, 750.er 10, 20 and 60 and 950. The TNFa INH activity was measured aft minutes and, by comparison with untreated samples, the percentage of TNFa INH activity was calculated according to formula (I). The results shown in Table 2 below demonstrate that the TNFa INH activity decreases in a time- and temperature-dependent manner.

28 Heat inactivation Percentage of Temperature (OC) Time (min) TNFa INH activity 100 56 20 100 93 60 20 26 15 27 20 10 13 b) Susceptibility to Trypsin Digestion Trypsin (500Lg) (Sigma, St. Louis, MO) in 0.2M Tris-HC1 buffer (pH 8.0) containing lmM calcium chloride was added to the pooled fractions of Sephacryl S-200 purified urinary TNFa INH of Example 2 and incubated at 370C for 4 hours. Another measure of trypsin (500ig) was added and digestion continued for a further 20 hours, at which time the reaction was terminated by addition of soybean trypsin inhibitor (2mg) (Sigma, St. Louis, MO). The percentage of TNFa INH inhibitory activity of the trypsin digest and the control was determined at a 1:20 final dilution of the pool of bulked fractions on L929 cells stimulated by rhTNFa in the presence of actinomycin D, according to formula (I). The results are shown in Table 3 below.

1 1 TrypAin inactivation Pecentage of TNFa INH OD 570nm Conditions activity Buffer alone 0 0.71 Trypsin + soybean trypsin inhibitor in 0 0.70 buffer Partially purified Sephadex S-200 urine 61 1.46 Partially purified Sephadex S-200 urine 23 1.03 digested by trypsin c) Treatment with Urea The Sephacryl S-200 purified TNFa INH of Example 2 was adjusted to 2M urea and extensively dialysed at 41P against phosphate buffered saline (PBS) containing 2M urea. Dialysis against PBS was -repeated prior to the bioassay. TNFa INH activity was found to be unaffected which indicates that inhibitory activity is not mediated by a molecule of low molecular weight bound to a protein.

Example 10 Demonstration of Competitive Inhibition The Sephacryl S-200 purified TNFa INH of Example 2 was tested at a 1:10 dilution against increasing amounts of rhTNFa on L929 cells An inverse correlation between the amount of rhTNFa present in the assay'and the degree of inhibition was observed (see Figure 3). Thus, the inhibitory activity is competitively overcome by increasing concentrations of rhTNFa.

Example 11 Inhibition of Wa-Mediated PGEq Production by Dermal Fibroblasts Human dermal fibroblasts were seeded at a concentration of 2. 0 x 104 cells/well and cultured for 48 hours. Cells were then stimulated with DMEM buffer supplemented with 10% FCS as a control. Cells were also stimulated with rhTNFi at concentrations ranging from 0.5 to 5mg/ml, and the effect of the TNFa INH from Example 5 was studied at three dilutions (1:20, 1:50 and 1:80) in the above buffer. After 72 hours of incubation, PGE2 production was measured in the supernatants by radioimmunoassay using a PGE 2 antiserum [see J M Dayer et al., J. Clin. Invest., 67, p1386 (1979)].

The results are shown in Table 4, below, and demonstrate that the ability of rhTNFa to stimulate PGE2 production by dermal fibroblasts was inhibited by the addition of TNFa INH at all three dilutions. At 1:80 dilution of Wa INH the inhibitory activity was partially overcome by increasing rhTNFa concentrations.

Concentration of rhTNF on human fibroblasts (pg/ml) PGE2 roduction by human dermal fibroblasts (ng/ml) Dilution of TNF INH on fibroblasts none 1:80 1:50 1:2C.

0 500 2,000 5,000 50.6 + 7.4 88.6 + 5.6 103.0 + 8.9 111.0 + 9.4 160.0 + 14.1 126.0 + 9.3 115.9 + 6.6 113.9 +7.1 331.7 + 28.4 217.2 + 10.7 156.7 + 10.7 115.3 + 21.3 381.7 + 19.6 257.2 + 13.7 253.1 + 21.2 221.6 + 16.0 1 1 1 Three different experiments were carried out with the same strain of fibroblasts. Buffer or TNFcc 1NH was incubated at various dilutions in the presence or absence of various concentrations of rhTNFa. PGE2 production by cultured human dermal fibroblasts was measured after three days. Values represent triplicate means of the three cultures + SEM (N=3).

1 31 Example 12 rhTNFa Binding Inhibition Assay Recombinant human Wa was iodinated by using the iodogen method of Fraker and Speck jr., Biochem. Biophys. Res. Comm., 80, p849 (1978). The specific activity of [125II-TNFa was 2.2x104 cpm/ng and produced a single band with a molecular weigh-l'.of 17 kDa when analysed by SDS PAGE. The human macrophage cell line U937 in aliquots of 106 Cells was cultured at 40 for 2 hours in a culture medium (200pil) comprising 10 RPMI 1640 (Gibco, Paisley, Scotland) supplemented with streptomycin (100pig/ml, penicillin (10OU/ml), 1.0%] glutamine and 10% foetal calf serum, and additionally containing 0.04% sodium azide and 0.5ng [123 I]- TNFa. Binding inhibition was performed by the addition of various dilutions of TNFA INH (1:20, 1:200 and 1;2000).

Non-specific binding was measured in the presence of a 100-fold excess of unlabelled rhTNFa, and free readioactivity was separated from the bound [123 I]-TNFa by centrifugation through an oil mixture as described by Robb et al., J. Exp. Med., 154, p1455 (1981). Cell bound [123 I]-TNR was measured in a gamma counter (LKB, Bromma, Sweden), and the percentage of binding inhibition was determined according to formula (II) Percentage of binding inhibition X 1 - epm with TNFx INH cpm of total binding - cpm non-specific binding cnm non-snecific bindinn - 32 Example 13

Effect of TNE INH on [125 I]-TWa Binding to U937 Cells U937 cells were preincubated for 1 hour at 200 in the culture medium of Example 12, in the presence of either [12SI]-TNFa alone or [1251]-TNFa with a 100-fold excess of unlabelled rhTNFa. The U937 cells were then washed with phosphate buffered saline (3 x 50m1) at 4 0. The cells incubated in [125I]-TNFa alone were divided into four batches and incubated with TNFa INH from Example 5 (1:20, 1:200 and 1:2000 dilutions) and with buffer alone, respectively.

23 The specific binding of 1 1 ---TNFato U937 cells was found to be inhibited at 40 by 1001%, 801% and 3510 by the three dilutions of Wa INH, 1:20, 1:200 and 1:2000, respectively (see Figure 5). The control batch which lacked TNFa INH showed no inhibitory activity. The binding inhibition of the two weaker dilutions was found to be increased to 90% and 60% when E125II-TNFu was preincubated with TNFa INH at dilutions 1:200 and 1:2000, respectively, prior to cell addition.

The experiment was repeated using the cells preincubated in the presence of [125 I]-TNFa and a 100-fold excess of unlabelled TNFa so that the percentage of binding inhibition could be corrected for nonspecific binding.

2 5, Example 14 Dissociation of a Pre-Formed TWa:U937 Complex U937 cells were preincubated for 1 hour in the presence of [123 I]-TWa as described in Example 12. The cells were washed and incubated at either 40 or 370 in the presence or absence of TNFa INH - 33 from Example 5. Cell-surface bound [j.2E-II-TNFa was found to dissociate faster in the presence of TFa INH than in its absence and this was found to occur in a time- and temperature-dependent manner (see Figure 6).

Example 15 Demonstration that TNFa INH is not Pro-ttt,.i-ytic foi41, hrTNFa [125 I]-TNFa was incubated at 20 0 for 1 hour in the presence of 3 different dilutions of TNR INH (1:20, 1:200 and 1:2000) and in the presence of buffer alone. When analysed by SDS PAGE and autoradiography the [125II-TNFa was found to migrate as a single band both in the absence and presence of TNFa INH showing the inhibitor to have no proteolytic effect.

Examole 16 Effect of TNFa INH on IL-1 Receptor Bindino Activity The activity of TNFa INH from Example 5 was tested in the IL-1/LAF (lymphocyte activating factor) assay when induced by IL-la or IL-lp [this assay is described by P. Seckinger et al., J. Immunol., 139, p1541 (1987) for an IL-I inhibitor protein]. A dose response o [3HI-thymidine incorporation (corresponding to thymocyte proliferation) was observed in up to 200pg/ml concentrations of both IL-la and IL-1p. Addition of TNR INH at levels observed to inhibit rhTNFa did not have any significant effect on the IL-1-induced thymocyte proliferation, proving inhibition to be specific for T%Vra only.

- 34 The results obtained are illustrated by reference to the accompanying drawings, in which:- - Figure 1 - shows the urinary TNFa INH activity profile of Sephacryl S-200 gel filtration. Column fractions.(lml) were tested at 1:10 dilution for effect in the rhTNFa (1.Ong/ml) cytotoxicity assay in the presence of actinomycin D (1.Opg/ml) (a---o). The line ( -) represents OD280nm of the fractions. Bars represent cell lysis measured by dye uptake in response to actinomycin D (0) and to actinomycin D plus hrTNFa (2) without urine. The molecular weight markers are dextran blue (DB), bovine serum albumin (BSA), ovelbumin (OA), a- chymotrypsinogen (aCT), ribonucle-ase A (RNase) and phenol red 0 -r e d).

Figure 2 - shows the urinary TNFa INH activity profile of chromatofocussing on a Mono-P column. Column fractions (lml) were tested at 1:10 dilution for effect in the rhTNFa (0.2ng/m1) cytotoxicity essay in the presence of actinomycin D (1.0 pg/ml) (o--o). The line represents OD28onm of the fractions, and ( -----) represents their pH. The bars are as described for Figure 1.

Figure 3 - shows the reversibility of TNFa INH activity. Open circles (o---o) represent OD57onm measured in the presence of actinomycin D, rhTNFa with TNFa INH; closed circles (---) represent OR 7onm measured in the presence of actinomycin and rhTNFa only and the bar (2) represents ODS7onm in the presence of "actinoijlycin D (1.Ogg/ml) alone.

- '35 Figure 4 - shows the elution profile of Sepacryl S-200 gel filtration with purified TNFa INH from Example 5. Column fractions (9m1) were sterilized and tested at 1:50 dilution against rhTNFa (1.Omg/ml) in the presence of actinomYcin D (1.0ig/ml) in the presence of actinomycin D (1. 0ig/ml) in the L929 cytotoxicity assay (o--o). The line (_) represents OD2..nm of the fractions. The bars are as defined for Figure 1.

Figure 5 - shows the SDS PACE analysis of purified TNFi INH of Example 7. SDS PAGE was performed as described by U. Laemmli et al., Nature, 277, loc 2-it. Samples were loaded onto 15% polyacrylamide gel with a 3'10 stacking gel and gels were silver-stained as described by C. Merril et al. , Proc. Natl. Acad. Sci. USA, 76, p4335 (1979). Samples run under nonreducing conditions were tested for biological activity by cutting 2mm slices from the gel and eluting the proteins by overnight incubation in lOmM Tris-HC1 pH 7.4 containing 2mM EDTA (total volume 200Ll). Fractions were tested at 1:10 dilution on L929 cells in the presence of rhTNFa (0. 15ng/ml).

Figure 6 - shows the effect of TNR INH on [125 I]-TWa binding to U937 cells. TNFa INH was incubated at three different dilutions in the presence of [123 I]-TWa with the U937 cell line as described in Example 13. Open squares (0 ------ F]) represent incubation in the presence of TNFa INH and the open triangles (A__---A) represent the control. Closed symbols refer to a 30 minute pre-incubation of TNFa INH at 200 with [123 I]-TWa in the culture medium prior to cell addition.

Figure 7 - shows the dissociation of a pre-formed TNFa: U937 complex. U937 cells were pre-incubated with [125I]-TNFa, washed and incubated with Wa INH as described in Example 14, at either 4 0 o r 3 7 0 At the time indicated, cell associated radioactivity was measured and percentage specific binding determined. On the graph, the value obtained from the control without the inhibitor has been substracted from the values obtained at the two temperatures, thus, 1001% corresponds to the value obtained without the addition of TNFa INH.

t.

c 1 37

Claims (25)

1 - A protein which inhibits tumour necrosis factor (TNF) a-mediated activity but does not block other proteins which.have in common with TNF certain but not all of the biological activities of TNF.

2. A protein which selectively inhibits tumour necrosis factor amediated activity and having one or more of the following characteristics:

(a) a molecular weight in the range 40 to 60 kDa, determined by molecular sieve chromatography; (b) an iso-electric point (pl) in the range 5.5 to 6-1, determined by chromatofocussing; (c) inhibition of the standard TNF assay of differential cytotoxicity for murine L929 cells treated with actinomycin D; (d) inhibition of TNF-induced PGE2' release from human fibroblasts-and synovial cells; (e) the inhibitor interferes with the binding of TNFa to U937 cells (a monocytic tumour line) as evidenced by inhibition of binding of radiolabelled TNFa (125I-TNFa); (f) the dissociation of a pre-formed TNFa: U937 cell complex is promoted by the inhibitor in a temperature dependent manner; (g) the inhibitor does not degrade MF by proteolytic cleavage; (h) the inhibitor does not inhibit IL-1 receptor-binding activity e.g. the binding of radiolabelled IL-1 (125I-IL-1a) to the murine thymoma subline EL4-6.1.

3. A protein which selectively inhibits tumour necrosis factor amediated activity and having one or more of the following characteristics (a) a molecular weight of about 33 kDa,-determined by SDS PAGE; (b) an iso-electric point (pI) in the range 5.5 to 6-1, determined by chromatofocussing; (c) inhibition of the standard TNF assay of differential cytotoxicity for murine L929 cells treated with actinomycin D; (d) inhibition of TNF-induced PGE2 release from human fibroblasts and synovial cells; (e) the inhibitor interferes with the binding of TNFa to U937 cells (a monocytic tumour line) as evidenced by inhibition of binding of radiolabel-led TNFa (1251-TNFa); (f) the dissociation of a pre-formed TNFa: U937 cell complex is promoted by the inhibitor in a temperature dependent manner; (9) the inhibitor does not degrade TNF by proteolytic cleavage; (h) the inhibitor does not inhibit IL-1 receptor-binding activity e.g. the binding of radiolabelled IL-1 (1251-IL-1a) to the murine thymoma subline EL4-6.1.

4. A protein as claimed- in either of claims 2 or 3 having the properties (a) and (b) together with one or more of the properties (c) to (h).

39

5. A protein as claimed in either of claims 2 or 3 having all of the properties (a) to (h). -

6. A protein according to any one of claims 1 to 5 which corresponds to a naturally occurring TNFa inhibitor.

7. A protein according to any one of claims 1 to 6 having an aminc terminal amino acid sequence as follows:

Asp-Ser-Val-Cys-Pro-Gln-Lys-Tyr-Ile-His-Pro-Gln-Cys-Asn-Ser-lle.

8. A protein according to claim 7 having an amino terminal amino acid sequence as follows:

Asp -Se r-Va I-Cy s-Pr o-GI n-Gl y-Ly s -Tyr-Il P.-Ri s-Pr o-GI n-Cy s-As n-Se r-II eAsn-Ser-Thr-Lys.

9. A protein according to any one of claims 1 to 8 and in which the amino acid sequence contains one or more deletions, substitutions, insertions, inversions or additions of allelic origin or otherwise, the resulting sequence having at least 80% homology with the parent protein and retaining essentially the same biological properties as the parent protein.

10. A protein according to claim 9 having at least 90% homology with the parent protein.

11. A protein according to any one of claims 1 to 10 in a substantially homogeneous form.

12. A protein according to any one of claims I to 11 which is a recombinant protein.

13. A protein according to any one of claims 1 to 12 which is a glycosylated protein.

14. A protein according to any one of claims 1 to 12 which is in a substantially unglycosylated state.

15. An exogeneous DNA comprising a nucleotide sequence coding for a protein as defined in any one of claims 1 to 11.

16. A cDNA comprising a nucleottde sequence coding for a protein according to any one of claims 1 to 11.

17. A recombinant expression vector comprising DNA according to either of claims 15 or 16.

18. A host cell transformed with an expression vector according to claim 17.

- 41 1 1

19. A method of producing a TNFa INH protein which comprises culturing a cell according to claim 18 and isolating the TNFa INH protein.

20. A recombinant protein produced according to the method of claim 19.

21. A method for the preparation of a TNFa INH protein comprising th steps of (a) concentration of urine from febrile patients; (b) ammonium sulphate precipitation; (c) anion-exchange chomatography; (d) cation-exchange chromatography; (e) gel filtration; (f) affinity chromatography; and (g) reverse phase FP

22. A protein characterised in that it is substantially identical to the protein obtained by the method of claim 21.

e

23. A pharmaceutical formulation comprising a TNFa inhibitor as defined in any one of claims I to 14 or claim 22 or a pharmaceutically acceptable derivative thereof and a pharmaceutically acceptable carrier therefor.

42 -

24. A protein as defined in any one of claims 1 to 14 or claim 22 for use in therapy.

25. A pharmaceutical formulation for use in the- manufacture of a medicament for the treatment of conditions associated with excessive or unregulated TNFa production, wherein said formulation comprises a TNFa inhibitor as defined in any one of claims I to 14 or claim 22 or or a pharmaceutically acceptable derivative thereof.

Published 1989 atThe Patent Office, State House.6671 Higl,,Hcdt,,.)rixLer. donWCJR4TP. Further cOP't!s maYbe obtainedfromThe PatentOffice. Sales Branch, St Mary Cray, Orpington, Kent BF.5 3RD. Printed by Multiplex techniques Itd, St Mar7 Cray. Kent, Con. 1/87